DETAILED ACTION
The amendment filed on 09/25/2026 has been entered and fully considered. Claims 1-21 are pending, of which claims 1, 6-7, 12, 14-16 and 20-21 are amended.
Response to Amendment
In response to amendment, the examiner modifies rejection over the prior art established in the previous Office action.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1-5 and 8-9 is/are rejected under 35 U.S.C. 103 as being unpatentable over Liao et al. (PNAS, 2020, IDS) (Liao).
Regarding claim 1, Regarding limitation 1(a), providing a sample comprising a plurality of nucleic acid molecules, wherein the sample has a volume of less than 50 microliters, Liao teaches using a digital PCR reaction mixture containing nucleic acid template. For example, Liao teaches “absolute quantification of a 280-bp amplicon DNA from the Listeria monocytogenes genome” and serially diluting “template DNA” for CLEAR-dPCR. Liao further teaches that “the reaction mixes were added into the well on the MiCA and centrifuged,” and that “around 443,000 droplets were generated in 200-μL regular PCR microcentrifuge tubes with an average diameter of 41 μm.” (p. 25632). A 41 μm diameter spherical droplet has a volume of about 36 pL, and 443,000 such droplets correspond to about 16 μL total aqueous reaction mixture, which is less than 50 μL. This is consistent with Liao’s teaching that no observable liquid loss occurred during MiCA emulsification and that the aqueous phase is completely transformed into droplets.
Regarding limitation 1(b), generating a plurality of partitions comprising processing materials and the plurality of nucleic acid molecules of the sample, upon flowing the sample through a hole and into a container, wherein the container is coupled with a supporting body within a centrifuge during generation of the plurality of partitions, Liao teaches generating droplet partitions from the dPCR reaction mix. Liao’s Fig. 1 caption states that “droplet PCR samples are centrifuged in a swing-bucket rotor and jet through the through holes on MiCA plates, yielding massive droplets monodispersed in silicone oil.” Liao further teaches that the reaction mixes are added into the MiCA well and centrifuged for 4 minutes at 15,000 rcf to generate about 443,000 droplets in 200-μL PCR microcentrifuge tubes (page 25632, par 6). Thus, the droplet partitions include the PCR processing materials and nucleic acid molecules, and are generated by flowing/jetting the sample through holes into a PCR tube container supported in a centrifuge rotor.
Regarding limitation 1(c), amplifying the plurality of nucleic acid molecules or derivatives thereof within the container, Liao teaches that PCR is performed after droplet generation in the same tube (Fig. 1). Liao explains that CLEAR-dPCR completes “sample preparation, PCR, and readout all in one tube,” (abstract) and further states that “PCR reactions inside these CLEAR-ed picoliter droplets are not affected by the addition of betaine.” (page 2530, par 0). Therefore, Liao teaches amplifying the nucleic acid molecules or PCR derivatives within the droplet-containing PCR tube.
Regarding limitation 1(d), scanning the plurality of partitions in the container to detect signals corresponding to the plurality of nucleic acid molecules or derivatives thereof, Liao teaches in situ light-sheet scanning of the droplet partitions in the PCR tube (page 25630, par 1). Liao states that “[a] laser light sheet scans the droplets in the PCR tube in situ,” (Fig. 1) and that plane fluorescence images are sequentially acquired (Fig. 1). Liao also teaches dual-wavelength 3D imaging with FAM and HEX/VIC labeling, high-speed scanning of the whole tube, and identifying/counting positive droplets from the 3D image data (Fig. 1). Accordingly, Liao teaches scanning the droplet partitions in the container to detect fluorescent signals corresponding to amplified nucleic acid molecules.
Liao does not expressly disclose generating at least 1 million partitions in the specific working example. However, Liao teaches that the number of through-hole channels was increased from 7 to 37 “to compensate for the loss of droplet generation rate,” and that the reaction mix was centrifuged for 4 minutes at 15,000 rcf to generate around 443,000 droplets (p. 25632). Liao therefore demonstrates a droplet generation rate of about 110,750 droplets per minute. At that same rate, generating 1 million droplets would take about 9 minutes, which is still a short generation duration and well within the claimed method. Further, Liao teaches that low droplet number contributes to uncertainty and that “nearly half a million droplets” allows CLEAR-dPCR to maintain reasonable uncertainty with over 10⁶ target molecules and expand the dynamic range of dPCR (p. 25630). Therefore, it would have been obvious to one of ordinary skill in the art to generate at least 1 million partitions in Liao by increasing the centrifugation/generation duration and/or the number of through-hole channels, because Liao teaches that droplet-generation throughput is adjustable and that increasing droplet number improves dynamic range and quantitative accuracy.
Regarding claim 2, As discussed above with respect to claim 1, Liao teaches generating droplet partitions by centrifuging a dPCR sample through a MiCA plate into a PCR tube. Liao further teaches that the sample flows through a plurality of holes, because Liao’s Fig. 1 states that “[d]roplet PCR samples are centrifuged in a swing-bucket rotor and jet through the through holes on MiCA plates, yielding massive droplets monodispersed in silicone oil.” Liao also expressly states in the Methods section that the number of “through-hole channels” was increased “from 7 to 37,” and that the reaction mixes were added into the MiCA well and centrifuged to generate droplets (page 25632, par 6).
Therefore, Liao teaches that step (b) comprises flowing the sample through a plurality of holes, and the “hole” recited in claim 1 is one of the plurality of through holes. Accordingly, Liao anticipates claim 2.
Regarding claim 3, As discussed above, Liao teaches a CLEAR-dPCR method for nucleic-acid quantification. Liao expressly teaches sequence-specific nucleic acid quantification and states that, for one benchmark assay, it performed “absolute quantification of a 280-bp amplicon DNA from the Listeria monocytogenes genome” using serially diluted “template DNA.” (page 25630, par 2). Liao also discusses “target molecule distribution” and maintaining reasonable uncertainty with over 10⁶ target molecules (page 25630, par 3). Therefore, Liao teaches that the plurality of nucleic acid molecules comprises a target nucleic acid molecule, as recited in claim 3.
Regarding claim 4, Liao further teaches that the target nucleic acid molecule can be a viral nucleic acid molecule. In particular, Liao states that its lossless CLEAR-dPCR pipeline addresses dPCR sensitivity issues in applications requiring high accuracy and low error rates, “such as the SARS-CoV-2 viral nucleic acids detection during the COVID-19 pandemic.” SARS-CoV-2 is a virus, and SARS-CoV-2 nucleic acids are viral nucleic acid molecules (page a5632, par 0). Therefore, Liao teaches that the target nucleic acid molecule comprises a viral nucleic acid molecule, as recited in claim 4.
Regarding claim 5, As discussed above with respect to claim 1, Liao teaches generating the droplet partitions by centrifugation. Liao further teaches that “[d]roplet PCR samples are centrifuged in a swing-bucket rotor and jet through the through holes on MiCA plates, yielding massive droplets monodispersed in silicone oil.” (Fig. 1). The swing-bucket rotor supports the PCR tube/container during centrifugation and is positioned within the centrifuge. Thus, Liao teaches that the supporting body comprises a swing bucket positioned within the centrifuge, as recited in claim 5.
Regarding claim 8, Liao teaches that wherein the plurality of partitions comprises a plurality of droplets, wherein a droplet of the plurality of droplets is surrounded by an immiscible fluid (silicon oil) (Fig. 1A).
Regarding claim 9, Liao teaches that the number of through-hole channels was increased from 7 to 37 “to compensate for the loss of droplet generation rate,” and that the reaction mix was centrifuged for 4 minutes at 15,000 rcf to generate around 443,000 droplets (p. 25632). Liao therefore demonstrates a droplet generation rate of about 110,750 droplets per minute. At that same rate, generating 1 million droplets would take about 9 minutes, which is still within a duration of 50 minutes.
Claim 6, 12-14, 16 and 20-21 is rejected under 35 U.S.C. 103 as being unpatentable over Liao et al. (PNAS, 2020, IDS) (Liao) in view of Colston et al. (US 9,156,010) (Colston).
Regarding claim 6, As discussed above, Liao teaches the method of claim 1. However, Liao does not expressly teach that the container is a well of a 96-well plate. Liao teaches generating the droplet emulsion in a PCR tube, where droplet PCR samples are centrifuged in a swing-bucket rotor and jet through MiCA through-holes to yield droplets in silicone oil, followed by in-tube PCR and light-sheet readout (Fig. 1).
Colston teaches droplet-based assay systems involving partitioning sample components into droplets, amplifying or reacting components within droplets, detecting the amplified components, and analyzing the resulting data (abstract). Colston further teaches that an emulsion may be transferred to “a sealable well/chamber of a plate (e.g., a 96-well PCR plate)” for thermal cycling (col. 37, lines 19-23). Colston also teaches that droplet generators and/or wells may correspond to the spacing, number, and arrangement of a standard microplate, including a microplate with 96 wells, and that such an arrangement permits use with instruments designed for standard microplates (col. 37, lines 44-47).
It would have been obvious to one of ordinary skill in the art before the effective filing date to modify Liao’s method by using a well of a 96-well PCR plate as the container for the generated droplet partitions. One of ordinary skill would have been motivated to make the modification because Colston teaches that droplet emulsions for nucleic-acid amplification may be contained in a sealable well/chamber of a 96-well PCR plate for thermal cycling, and further teaches that a 96-well microplate arrangement enables compatibility with standard microplate instruments. The modification would have been the predictable use of a known PCR-compatible container format to increase parallel sample handling and throughput in Liao’s dPCR workflow.
Regarding claim 12, Liao teaches a method comprising providing a sample comprising a plurality of nucleic acid molecules. Liao teaches CLEAR-dPCR for “sequence-specific nucleic acid quantification” (page 25628, par 1) and teaches performing absolute quantification of a 280-bp amplicon DNA from the Listeria monocytogenes genome using serially diluted template DNA. (page 25630, par 2).
Liao further teaches that the sample has a volume of less than 50 microliters. Liao teaches that the reaction mixes were added into the well on the MiCA and centrifuged to generate around 443,000 droplets in 200-µL regular PCR microcentrifuge tubes, with an average droplet diameter of 41 µm (page 25632, par 6). A 41 µm diameter droplet has a volume of about 36 pL, and 443,000 droplets correspond to about 16 µL of aqueous reaction mixture, which is less than 50 µL.
Liao teaches generating a plurality of partitions comprising processing materials and the plurality of nucleic acid molecules of the sample upon flowing the sample through a hole and into a container (Fig. 1). Specifically, Liao teaches generating an optically transparent dPCR emulsion by dispersing an aqueous dPCR mix using MiCA centrifugation, and Fig. 1 states that “[d]roplet PCR samples are centrifuged in a swing-bucket rotor and jet through the through holes on MiCA plates, yielding massive droplets monodispersed in silicone oil.” The dPCR mix includes PCR processing materials, and the droplets are partitions containing the nucleic acid molecules.
Liao teaches that the container is coupled with a supporting body within a centrifuge during generation of the plurality of partitions. Liao’s Fig. 1 expressly shows and describes droplet PCR samples centrifuged in a swing-bucket rotor, and Liao’s Methods state that the reaction mixes were centrifuged for 4 minutes at 15,000 rcf.
Liao teaches or suggests that the plurality of partitions comprises at least 500,000 partitions. Liao states in the abstract that light-sheet fluorescence microscopy was used “to acquire a three-dimensional (3D) image of a half million droplets sealed in a tube in seconds,” and later explains that CLEAR-dPCR operates with “nearly half a million droplets.” To the extent Liao’s specific example of “around 443,000 droplets” is considered slightly below 500,000, it would have been obvious to generate at least 500,000 droplets because Liao expressly teaches increasing the number of through-hole channels from 7 to 37 to compensate for droplet-generation rate and teaches that higher droplet number improves dynamic range and reduces uncertainty. Increasing the number of droplets from about 443,000/nearly half a million to at least 500,000 would have been a routine optimization of a result-effective variable, particularly because 500,000 droplets of 41 µm diameter would still require only about 18 µL of aqueous sample, which remains below the claimed 50 µL limit.
Liao does not expressly disclose generating at least 1 million partitions in the specific working example. However, Liao teaches that the number of through-hole channels was increased from 7 to 37 “to compensate for the loss of droplet generation rate,” and that the reaction mix was centrifuged for 4 minutes at 15,000 rcf to generate around 443,000 droplets (p. 25632). Liao therefore demonstrates a droplet generation rate of about 110,750 droplets per minute. At that same rate, generating 1 million droplets would take about 9 minutes, which is still a short generation duration and well within the claim 9 claimed method. Further, Liao teaches that low droplet number contributes to uncertainty and that “nearly half a million droplets” allows CLEAR-dPCR to maintain reasonable uncertainty with over 10⁶ target molecules and expand the dynamic range of dPCR. Liao 2020 Therefore, it would have been obvious to one of ordinary skill in the art to generate at least 1 million partitions in Liao by increasing the centrifugation/generation duration and/or the number of through-hole channels, because Liao teaches that droplet-generation throughput is adjustable and that increasing droplet number improves dynamic range and quantitative accuracy.
Again, Liao does not expressly teach that the container is a well of a 96-well plate.
Colston teaches droplet-based assay systems involving partitioning sample components into droplets, amplifying or reacting components within droplets, detecting the amplified components, and analyzing the resulting data (abstract). Colston further teaches that each emulsion may be transferred to “a sealable well/chamber of a plate (e.g., a 96-well PCR plate)” for thermal cycling (col. 37, lines 19-23). Colston also teaches that a plate may define an array of wells/chambers for holding emulsions and that droplet signals may be detected while the emulsions remain in a plate, well, or vial (col. 35, line 11-35).
It would have been obvious to modify Liao’s method by using a well of a 96-well PCR plate as the container for the generated droplet partitions, as taught by Colston. Liao teaches a dPCR workflow using centrifugation-based droplet generation, PCR, and signal readout, while Colston teaches using wells of a 96-well PCR plate as PCR-compatible containers for droplet emulsions. One of ordinary skill in the art would have been motivated to use the known 96-well PCR plate format to increase parallel sample handling, improve compatibility with standard PCR and plate-based laboratory workflows, and enable multiple dPCR samples to be processed in an array format. The modification would have involved substituting one known PCR-compatible container format for another with predictable results.
Regarding claim 13, As discussed above with respect to claim 12, Liao teaches generating droplet partitions by centrifuging a dPCR sample through a MiCA plate into a PCR tube. Liao further teaches that the sample flows through a plurality of holes, because Liao’s Fig. 1 states that “[d]roplet PCR samples are centrifuged in a swing-bucket rotor and jet through the through holes on MiCA plates, yielding massive droplets monodispersed in silicone oil.” Liao also expressly states in the Methods section that the number of “through-hole channels” was increased “from 7 to 37,” and that the reaction mixes were added into the MiCA well and centrifuged to generate droplets (page 25632, par 6).
Regarding claim 14, As discussed above with respect to claim 12, Liao in view of Colston teaches the method of claim 12. Liao further teaches amplifying the plurality of nucleic acid molecules or derivatives thereof within the well. Liao teaches that CLEAR-dPCR completes “dPCR sample preparation, PCR, and readout all in one tube” (abstract) and further teaches that “[a]fter PCR, in situ noninvasive and high-throughput readout of whole-tube emulsion is accomplished” by light-sheet fluorescence imaging (page 25628, par 4). Liao also explains that “PCR reactions inside these CLEAR-ed picoliter droplets are not affected by the addition of betaine,” (page 25630, par 0), confirming amplification within the droplet partitions in the PCR tube.
Thus, Liao teaches amplifying the plurality of nucleic acid molecules or derivatives thereof within the well, as recited in claim 14.
Regarding claim 16, As discussed above with respect to claim 12, Liao in view of Colston teaches the method of claim 12. Liao further teaches scanning the plurality of partitions to detect signals corresponding to the plurality of nucleic acid molecules or derivatives thereof (Fig. 1).
Specifically, Liao teaches in situ optical readout of the droplet partitions after PCR (Fig. 1). Liao explains that it built a light-sheet fluorescence microscope “to noninvasively read the fluorescent signals of massive droplets that stack inside the sealed PCR tube,” and that the droplets were scanned across a laser light sheet while fluorescence images were collected (page 25632, par 7). Liao further teaches that the fluorescence distribution of the entire emulsion was reconstructed (page 25632, par 7), and that positive droplets were identified and counted to obtain nucleic acid copy number and concentration (page 25632, par 8).
Liao also states that Fig. 3 shows “the light-sheet fluorescence reader and droplet counting,” including signal/background measurement in a raw CLEAR-dPCR image and 3D reconstruction in which fluorescently positive droplets are located and counted.
Thus, Liao teaches scanning the plurality of partitions to detect signals corresponding to the plurality of nucleic acid molecules or derivatives thereof, as recited in claim 16.
Regarding claim 20, Liao further teaches that the target nucleic acid molecule can be a viral nucleic acid molecule. In particular, Liao states that its lossless CLEAR-dPCR pipeline addresses dPCR sensitivity issues in applications requiring high accuracy and low error rates, “such as the SARS-CoV-2 viral nucleic acids detection during the COVID-19 pandemic.” SARS-CoV-2 is a virus, and SARS-CoV-2 nucleic acids are viral nucleic acid molecules (page a5632, par 0). Therefore, Liao teaches that the target nucleic acid molecule comprises a viral nucleic acid molecule, as recited in claim 20.
Regarding claim 21, As discussed above with respect to claim 13, Liao teaches generating the droplet partitions by centrifugation. Liao further teaches that “[d]roplet PCR samples are centrifuged in a swing-bucket rotor and jet through the through holes on MiCA plates, yielding massive droplets monodispersed in silicone oil.” (Fig. 1). The swing-bucket rotor supports the PCR tube/container during centrifugation and is positioned within the centrifuge. Thus, Liao teaches that the supporting body comprises a swing bucket positioned within the centrifuge, as recited in claim 21.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Liao et al. (PNAS, 2020, IDS) (Liao) in view of Shin et al. (Sensors & Actuators: B. Chemical, 2019) (Shin).
Regarding claim 7, As discussed above with respect to claim 1, Liao teaches the method of claim 1. Liao teaches that its CLEAR-dPCR method uses “centrifugation-based droplet generation,” optical clearing of PCR emulsion, and in situ counting by light-sheet fluorescence microscopy (p. 25628). Liao further teaches that droplet PCR samples are centrifuged in a swing-bucket rotor and jet through through-holes on MiCA plates, yielding massive droplets monodispersed in silicone oil (Fig. 1a, p. 25629).
Liao does not expressly teach that the plurality of partitions comprises at least 2 million partitions. However, Liao teaches that increasing droplet number improves digital PCR performance. In particular, Liao explains that low droplet number contributes to higher uncertainty, and that “nearly half a million droplets” allows CLEAR-dPCR to maintain reasonable uncertainty and expand the dynamic range of dPCR (p. 25630). Liao 2020 Liao also teaches that its example increased the number of through-hole channels from 7 to 37 to compensate for droplet-generation rate and generated around 443,000 droplets in 4 minutes (p. 25632).
Shin teaches centrifuge-based generation of smaller picoliter droplets. Shin teaches a “centrifuge-based step emulsification device” housed in a microtube for simple and fast generation of monodisperse picoliter droplets (abstract). Shin teaches that centrifugation using a commercial centrifuge drives the dispersed phase through a microchannel, pinches off droplets, and transports the droplets to the bottom of the microtube by centrifugal force (p. 1-2). Shin further teaches water-in-oil picoliter droplets with diameters ranging from 18 to 90 µm (abstract). Shin specifically teaches typical droplet-generation conditions of 40 µL of water and 3 minutes of centrifugation, and reports tens of thousands of monodisperse droplets having an average diameter of 23.1 µm after a single centrifuge operation (p. 3). Shin also teaches that droplet diameter is controllable by microchannel height, including monodisperse droplets of 22 µm, 63 µm, and 88 µm (p. 4).
It would have been obvious to one of ordinary skill in the art to modify Liao’s centrifuge-based CLEAR-dPCR droplet-generation method to use smaller centrifuge-generated picoliter droplets, as taught by Shin, and to increase the number of generated droplets, in order to increase the number of digital PCR partitions while maintaining a sample volume below 50 µL and improving digital PCR dynamic range and quantification accuracy. The modification would have had a reasonable expectation of success because both Liao and Shin use centrifugation to generate water-in-oil droplets, and Shin demonstrates that centrifuge-based devices can produce smaller, monodisperse picoliter droplets suitable for biochemical reaction compartments.
For example, 2 million spherical droplets having Shin’s 23.1 µm average diameter would correspond to about 13 µL of aqueous volume, which is below the less-than-50-µL sample volume recited in claim 1. Thus, using Shin’s smaller centrifuge-generated picoliter droplets would have predictably allowed Liao’s centrifuge-based dPCR workflow to generate at least 2 million droplet partitions within the claimed sample-volume range.
Accordingly, Liao in view of Shin teaches or suggests the method of claim 1, wherein the plurality of partitions comprises at least 2 million partitions, as recited in claim 7.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Liao et al. (PNAS, 2020, IDS) (Liao) in view of Jiang et al. (Scientific Reports, 2020) (Jiang).
Regarding claim 10, As discussed above with respect to claim 1, Liao teaches scanning the plurality of partitions in the container to detect signals corresponding to nucleic acid molecules or derivatives thereof. Liao teaches that “[a] laser light sheet scans the droplets in the PCR tube in situ,” (Fig. 1B) and that the system performs dual-channel fluorescence imaging using FAM and HEX/VIC labeling (Fig. 1C). However, Liao does not expressly teach scanning across at least 4 color channels.
Jiang teaches a digital PCR system and assay using at least 4 color channels. Jiang teaches “a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels,” (abstract) and further teaches a quadruplex assay that quantifies multiple nucleic-acid targets in the same dPCR assay. Jiang specifically teaches using four fluorophores, FAM, VIC, TYE665, and TAMRA, to identify SMN1, SMN2, total SMN, and RPPH1, respectively (page 2, par 2). Jiang also teaches that the entire partition arrays are imaged before and after PCR “with up to five optical channels.” (page 9, par 1).
It would have been obvious to one of ordinary skill in the art to modify Liao’s dPCR scanning method to scan the partitions across at least 4 color channels, as taught by Jiang. Liao already teaches fluorescent dPCR scanning of droplet partitions using multiple channels, and Jiang teaches that higher-order multiplex dPCR using four fluorophores/five optical channels permits simultaneous detection and quantification of multiple nucleic-acid targets and an internal reference in a single assay. A person of ordinary skill would have been motivated to make the modification to increase multiplexing capability and obtain more target information from the same dPCR sample, with a reasonable expectation of success because both references concern fluorescence-based digital PCR detection of nucleic-acid targets in partitioned reaction volumes.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Liao et al. (PNAS, 2020, IDS) (Liao) in view of Beck et al. (Cinical Chemistry, 2013) (Beck).
Regarding claim 11, As discussed above with respect to claim 1, Liao teaches scanning the plurality of partitions in the container to detect signals corresponding to nucleic acid molecules or derivatives thereof (Fig. 1). Liao teaches that CLEAR-dPCR performs “sample preparation, PCR, and readout all in one tube,” (abstract) and that a laser light sheet scans droplets in the PCR tube in situ to acquire fluorescence images and count positive droplets (Fig. 1B).
Liao does not expressly teach that the signals are analyzed as being indicative of rejection events of transplanted organs.
However, Beck teaches using droplet digital PCR to quantify graft-derived cell-free DNA in transplant recipients as a biomarker for graft injury and rejection (background). Beck teaches that cell-free DNA from grafts in the circulation of transplant recipients is a potential biomarker of rejection (background), and that Beck developed a rapid and cost-effective method using digital droplet PCR (page1732, par 4). Beck further teaches that plasma cfDNA was preamplified (page 1734, par 2), SNP assays were used, and graft-derived cfDNA was quantified using ddPCR (page 1734, par 3). Beck reports that, in two patients with biopsy-proven rejection, graft-derived cfDNA increased to greater than 60%, and concludes that the ddPCR technique is a promising biomarker for early detection of rejection (page 1732, par 4).
It would have been obvious to one of ordinary skill in the art to further analyze the signals generated from Liao’s scanned dPCR partitions to determine signals indicative of rejection events of transplanted organs, as taught by Beck. Liao already teaches a dPCR workflow for sensitive nucleic-acid quantification by partitioning, amplification, scanning, and counting positive droplets. Beck teaches that ddPCR quantification of graft-derived cfDNA from transplant recipients provides information indicative of transplant rejection. A person of ordinary skill would have been motivated to apply Beck’s transplant-rejection ddPCR analysis to Liao’s dPCR scanning/counting method to obtain a sensitive, quantitative, and noninvasive indication of graft injury or rejection using nucleic-acid signals from partitioned PCR reactions.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Liao et al. in view of Colston as applied to claims 6, 12-14, 16 and 20-21 above, and further in view of Schulz et al. (Molecules, 2020) (Schulz).
Regarding claim 15, As discussed above, Liao in view of Colston teaches the method of claim 14, including amplification of nucleic acid molecules within droplet partitions. Liao teaches PCR amplification, but does not expressly teach that the amplification comprises isothermal amplification.
However, Schulz teaches digital droplet amplification using centrifugal droplet generation and expressly teaches compatibility with digital droplet loop-mediated isothermal amplification (ddLAMP). (abstract). Schulz states that its system demonstrates “a digital droplet polymerase chain reaction (ddPCR) and digital droplet loop mediated isothermal amplification (ddLAMP),” (abstract) and that both applications show high linearity, demonstrating that the droplet generation method does not compromise assay performance (abstract). Schulz also states that biochemical compatibility was demonstrated with “two exemplary amplification reactions,” namely ddPCR and ddLAMP (page 2, par 3).
It would have been obvious to one of ordinary skill in the art to perform the amplification in Liao’s droplet-based digital nucleic-acid analysis using an isothermal amplification technique, such as ddLAMP as taught by Schulz. Liao teaches droplet-based digital nucleic-acid amplification and fluorescent detection, while Schulz teaches that digital droplet amplification is compatible with both PCR and loop-mediated isothermal amplification. A person of ordinary skill would have been motivated to use isothermal amplification to provide an alternative digital nucleic-acid amplification workflow that avoids thermal cycling and is compatible with droplet-based partitioned assays. The substitution would have had a reasonable expectation of success because Schulz expressly demonstrates ddLAMP in droplets.
Claim 17 and 19 is rejected under 35 U.S.C. 103 as being unpatentable over Liao et al. in view of Colston as applied to claims 6, 12-14, 16 and 20-21 above, and further in view of Madic et al. (Oncotarget, 2018) (Madic).
Regarding claim 17, As discussed above with respect to claims 12 and 16, Liao in view of Colston teaches the method of claim 16, including scanning the plurality of partitions to detect signals corresponding to nucleic acid molecules or derivatives thereof. Liao teaches dual-wavelength/dual-channel light-sheet fluorescence imaging using FAM and HEX/VIC labeling, but Liao does not expressly teach scanning across at least 6 color channels.
Madic teaches six-color digital PCR for detecting nucleic-acid targets. Madic is titled “EGFR C797S, EGFR T790M and EGFR sensitizing mutations in non-small cell lung cancer revealed by six-color crystal digital PCR”. Madic teaches that a 6-color digital PCR assay was developed for detecting and quantifying EGFR mutations, and that the assay used 6 fluorescence detection channels to differentiate multiple EGFR mutations in a single reaction (abstract). Madic further teaches that the six-color assay used detection channels including FAM, Atto700, Cyanine 5, Cyanine 3, and ROX for different EGFR targets, and that six-color digital PCR experiments were performed (page 37394, par 2).
Madic also teaches scanning/imaging droplet partitions across six detection channels (Fig. 4). In particular, Madic teaches that PCR reactions were loaded on Sapphire chips, “compartmentalized in four chambers into 2D monolayers of droplet partitions,” thermocycled, and imaged by fluorescence microscopy (page 37404, par 1). Madic further teaches that “[f]ilter sets” were selected for fluorescence readout in six distinct detection channels (page 37404, par 1).
It would have been obvious to one of ordinary skill in the art to modify Liao’s fluorescence-based dPCR scanning method to scan partitions across at least 6 color channels, as taught by Madic. Liao already teaches multiplex fluorescence dPCR detection of nucleic-acid targets from droplet partitions, and Madic teaches that using six fluorescence detection channels increases multiplexing power and allows multiple nucleic-acid targets/mutations to be differentiated in a single dPCR reaction. A person of ordinary skill would have been motivated to make the modification to increase the number of nucleic-acid targets detectable from the same partitioned sample, with a reasonable expectation of success because Madic expressly demonstrates six-color fluorescence readout of digital PCR droplet partitions.
Regarding claim 19, As discussed above with respect to claims 12 and 16, Liao in view of Colston teaches the method of claim 16, including scanning the plurality of partitions to detect signals corresponding to nucleic acid molecules or derivatives thereof. Liao teaches scanning droplet partitions in a PCR tube by light-sheet fluorescence imaging and counting fluorescently positive droplets corresponding to amplified nucleic acid targets.
Liao does not expressly teach that the detected signals are indicative of cancerous material of the sample. Madic teaches digital PCR detection of cancer-associated nucleic-acid mutations. Specifically, Madic teaches a six-color digital PCR assay for detecting and quantifying prevalent EGFR sensitizing and resistance mutations in non-small cell lung cancer (abstract). Madic further teaches evaluating the assay on tumor and plasma samples from NSCLC patients, and measuring EGFR mutant allelic fraction in those samples by digital PCR.
It would have been obvious to one of ordinary skill in the art to use Liao’s partitioned digital PCR scanning method to detect signals indicative of cancerous material, as taught by Madic. Liao teaches sensitive fluorescence-based digital PCR detection of nucleic-acid targets in droplet partitions, while Madic teaches that digital PCR signals from EGFR mutations are useful for detecting and monitoring cancer-associated material in tumor or plasma samples from NSCLC patients. A person of ordinary skill would have been motivated to apply Madic’s cancer-mutation detection assay to Liao’s dPCR partition/scanning workflow to sensitively detect cancer-associated nucleic acid material in a sample, with a reasonable expectation of success because both references use fluorescence-based digital PCR detection of nucleic-acid targets in partitioned reaction volumes.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Liao et al. in view of Colston as applied to claims 6, 12-14, 16 and 20-21 above, and further in view of Beck et al. (Cinical Chemistry, 2013) (Beck).
Regarding claim 18, Liao does not expressly teach that the signals are analyzed as being indicative of rejection events of transplanted organs.
However, Beck teaches using droplet digital PCR to quantify graft-derived cell-free DNA in transplant recipients as a biomarker for graft injury and rejection (background). Beck teaches that cell-free DNA from grafts in the circulation of transplant recipients is a potential biomarker of rejection (background), and that Beck developed a rapid and cost-effective method using digital droplet PCR (page1732, par 4). Beck further teaches that plasma cfDNA was preamplified (page 1734, par 2), SNP assays were used, and graft-derived cfDNA was quantified using ddPCR (page 1734, par 3). Beck reports that, in two patients with biopsy-proven rejection, graft-derived cfDNA increased to greater than 60%, and concludes that the ddPCR technique is a promising biomarker for early detection of rejection (page 1732, par 4).
It would have been obvious to one of ordinary skill in the art to further analyze the signals generated from Liao’s scanned dPCR partitions to determine signals indicative of rejection events of transplanted organs, as taught by Beck. Liao already teaches a dPCR workflow for sensitive nucleic-acid quantification by partitioning, amplification, scanning, and counting positive droplets. Beck teaches that ddPCR quantification of graft-derived cfDNA from transplant recipients provides information indicative of transplant rejection. A person of ordinary skill would have been motivated to apply Beck’s transplant-rejection ddPCR analysis to Liao’s dPCR scanning/counting method to obtain a sensitive, quantitative, and noninvasive indication of graft injury or rejection using nucleic-acid signals from partitioned PCR reactions.
Response to Arguments
Applicant’s arguments with respect to claim(s) 1 and 12 have been considered but are moot in view of new ground of rejection.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to XIAOYUN R XU, Ph. D. whose telephone number is (571)270-5560. The examiner can normally be reached M-F 8am-5pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Lyle Alexander can be reached at 571-272-1254. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/XIAOYUN R XU, Ph.D./Primary Examiner, Art Unit 1797
